Javier Plasencia

Plant Sphingolipids: Structure, Synthesis and Function

Sphingolipids are major structural components of endomembranes and dynamic regulators of basic cellular processes in plants. Advances during the past decade have revealed that sphingolipids are essential molecules in plants, and many of the genes for sphingolipid biosynthetic enzymes have been identified and characterized. In addition, improved methods for sphingolipid extraction and analysis have uncovered the immense structural complexity and quantitative importance of sphingolipids in plant cells. These advanced analytical methods have also been increasingly applied to the characterization of Arabidopsis thaliana mutants to provide unexpected insights into sphingolipid metabolism and function. Complementing these studies is a growing awareness that sphingolipids are one of the most abundant lipid components of the plasma membrane of plant cells and may play a role in the organization and function of membrane microdomains that are important for cell surface activities and for trafficking of proteins to the plasma membrane. Furthermore, sphingolipid metabolites including free and phosphorylated forms of long-chain bases and ceramides have been linked as bioactive regulators to a number of cellular processes (e.g., programmed cell death) that are important for abiotic stress resistance, plant development, and plant—pathogen interactions. This review provides a synopsis of the rapidly progressing field of plant sphingolipid biology and highlights gaps in our knowledge of the metabolism and function of these molecules in plants.

Aflatoxins in Maize: A Mexican Perspective

Aflatoxins are carcinogenic metabolites produced by Aspergillus flavus, a fungal pathogen that infects maize both in the field and during storage. Mexico is the center of origin of maize and its production in most parts in the country is characterized by the employment of a wide diversity of open‐pollinated genotypes adapted to certain environments. In most regions, maize is produced under rain fed conditions with low fertilizer and pesticide input and consequent low yields, probably fostering A. flavus infection in drought‐stressed plants. In addition, poor pest control increases insect damage, facilitating fungal infection and aflatoxin contamination. Ideally, management of aflatoxin contamination should begin with the employment of resistant genotypes as has been demonstrated by several U.S. breeding programs. However, in Mexico the wide genetic diversity of maize has not been fully exploited to identify resistance to aflatoxin contamination in breeding programs, thus impeding the reduction of aflatoxin levels in the field. Additional complications come from the fact that transgenic maize expressing insecticidal protein or any other trait to reduce aflatoxin is not viable in Mexico due to a government prohibition on the use of genetically modified maize. Maize is a staple crop in Mexico with high consumption in forms such as tortillas; thus, aflatoxin contamination is a significant threat to human health. Although aflatoxins are partially destroyed during the alkaline cooking procedure (called nixtamalization) to prepare tortillas, residual levels of aflatoxins might be considerable. Although important research has been conducted in several aspects of aflatoxin contamination of maize by universities, agricultural centers, and some government agencies, a full mycotoxin research program is needed in Mexico to ascertain the extents of aflatoxin contamination in different parts of the country and to develop economically viable technology to reduce aflatoxin exposure.

COMPARISON AMONG DNA POLYMERASES 1, 2 AND 3 FROM MAIZE EMBRYO AXES. A DNA PRIMASE ACTIVITY COPURIFIES WITH DNA POLYMERASE 2

Three DNA polymerase activities, named 1, 2 and 3 were purified from maize embryo axes and were compared in terms of ion requirements, optimal pH, temperature and KCl for activity, response to specific inhibitors and use of templates. All three enzymes require a divalent cation for activity, but main differences were observed in sensitivity to inhibitors and template usage: while DNA polymerases 1 and 2 were inhibited by N-ethyl maleimide and aphidicolin, inhibitors of replicative-type enzymes, DNA polymerase 3 was only marginally or not affected at all. In contrast, DNA polymerase 3 was highly inhibited by very low concentrations of ddTTP, an inhibitor of repair-type enzymes, and a 100-fold higher concentration of the drug was needed to inhibit DNA polymerases 1 and 2. Additionally, DNA polymerases 1 and 2 used equally or more efficiently the synthetic template polydA-oligodT, as compared to activated DNA, while polymerase 3 used it very poorly. Whereas DNA polymerases 1 and 2 shared properties of replicative-type enzymes, DNA polymerase 3 could be a repair-type enzyme. Moreover, a DNA primase activity copurified with the 8000-fold purified DNA polymerase 2, strenghtening the suggestion that polymerase 2 is a replicative enzyme, of the α-type. This DNA primase activity was also partially characterized. The results are discussed in terms of relevant data about other plant DNA polymerases and primases reported in the literature.

Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici

Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization.

The role of sphinganine analog mycotoxins on the virulence of plant pathogenic fungi.

Functional genomics and recent analytical developments have contributed to partially resolving the complexity and the important roles of sphingolipids in plant cell physiology. In the light of these discoveries, it is worthwhile to reassess the function of the sphinganine analog mycotoxins, which are produced by plant pathogenic fungi. The mode of action of sphinganine analog mycotoxins, which disrupt cellular sphingolipid metabolism, contributes to unravelling the role of sphingolipid intermediaries in the defense reaction mediated by the phytohormones salicylic acid, jasmonic acid, and ethylene. Thus toxin production appears to be an effective pathogen strategy to modulate the host cell defense response.

Deciphering the link between salicylic acid signaling and sphingolipid metabolism

The field of plant sphingolipid biology has evolved in recent years. Sphingolipids are abundant in cell membranes, and genetic analyses revealed essential roles for these lipids in plant growth, development, and responses to abiotic and biotic stress. Salicylic acid (SA) is a key signaling molecule that is required for induction of defense-related genes and rapid and localized cell death at the site of pathogen infection (hypersensitive response) during incompatible host–pathogen interactions. Conceivably, while levels of SA rapidly increase upon pathogen infection for defense activation, they must be tightly regulated during plant growth and development in the absence of pathogens. Genetic and biochemical evidence suggest that the sphingolipid intermediates, long-chain sphingoid bases, and ceramides, play a role in regulating SA accumulation in plant cells. However, how signals generated from the perturbation of these key sphingolipid intermediates are transduced into the activation of the SA pathway has long remained to be an interesting open question. At least four types of molecules – MAP kinase 6, reactive oxygen species, free calcium, and nitric oxide – could constitute a mechanistic link between sphingolipid metabolism and SA accumulation and signaling.

Cloning and characterization of a COBRA -like gene expressed de novo during maize germination

The search for germination-specific genes has been a laborious and unrewarding task, since many of the genes expressed during germination are also expressed in embryogenesis or in other developmental stages. By using mRNA differential display of transcript populations from maize (Zea mays L.) embryo axes, germinated for different times with or without a previous osmopriming treatment, a 682 bp cDNA was isolated that was present only after 24 h germination, and absent during osmopriming or during early germination. Screening of a cDNA library using the 682 bp probe yielded a 1554 bp cDNA that contained an open reading frame coding for 436 amino acids. This gene, referred to as ZmAA9-24, was expressed in root tissues, but was not detected in shoot or leaf tissues. Expression of ZmAA9-24 occurred earlier during germination (by 15 h) if embryo axes were imbibed in the presence of cytokinins or if seeds were previously osmoprimed. The predicted protein sequence of ZmAA9-24 is 39.6% identical to the product of the recently identified Arabidopsis gene COBRA (54.5% in the central region), which appears to participate in the regulation of cell expansion, particularly in roots, and belongs to the glycosylphosphatidylinositol (GPI)-anchored protein family. ZmAA9-24 expression might be regulated by both cell expansion and the cell cycle, processes that have a central role during seed germination.